Determination of Molecular Marker Expression Can Predict Clinical Outcome in Colon Carcinomas Gennaro Galizia, 1 Eva Lieto, 1 Francesca Ferraraccio, 2 Michele Orditura, 3 Ferdinando De Vita, 3 Paolo Castellano, 1 Vincenzo Imperatore, 1 Ciro Romano, 4 Fortunato Ciardiello, 3 Bruno Agostini, 2 and Carlo Pignatelli 1 Divisions of 1 Surgical Oncology, 2 Pathology, and 3 Medical Oncology, and 4 Division of Internal Medicine, Allergy and Clinical Immunology; “F. Magrassi and A. Lanzara” Department of Clinical and Experimental Medicine and Surgery; Second University of Naples School of Medicine, Naples, Italy ABSTRACT Purpose: Conventional staging procedures are often un- able to precisely predict prognosis in colorectal cancer (CRC). In this study, we set out to investigate the possible role of molecular/structural indicators involved in cell cycle regulation (p27 and p53), apoptosis (p53 and p27), and tumor neoangiogenesis [p53, vascular endothelial growth factor (VEGF), and microvessel count] in predicting tumor behavior and clinical outcome in CRC patients Experimental Design: Analysis of the above indicators was performed by immunohistochemistry on 104 CRC pa- tient samples and 25 normal colon mucosa specimens. Results: Intense p27 nuclear staining was found in nor- mal colon mucosa, with p53 nuclear staining and VEGF cytoplasmic accumulation <10%, and low microvessel count. In contrast, in CRC samples, p27 was down-regulated in 53.8%, p53 protein was overexpressed in 52%, and VEGF stained positive in 67.3% of the cases, respectively. Multiple regression analysis showed that molecular markers were strongly correlated. In patients treated with curative sur- gery, a significant relationship was seen between p27 down- regulation and Dukes’ stage, nodal status, and the presence of distant metastases. VEGF overexpression correlated sig- nificantly with Dukes’ stage, tumor (t) and metastasis (m) parameters, and left site. Stepwise regression selected p27, p53, VEGF, and Dukes’ stage as the best combination of variables capable of predicting both disease-specific and disease-free survival. Conclusions: The investigated indicators may be useful for the prediction of outcome and recurrence rate in cura- tively treated CRC patients. In conjunction with clinical and pathological staging, they may provide a stronger indication of clinical outcome than staging alone and help better select therapeutic options in CRC patients. INTRODUCTION Colorectal cancer (CRC) is one of the major causes of cancer death worldwide, accounting for more than 150,000 new cases and 55,000 deaths in the United States every year and 125,000 mortalities each year in Europe (1, 2). To date, radical surgery, followed by adjuvant chemotherapy when ap- propriate, is the mainstay of therapy for patients with localized disease (3– 6). However, despite adjuvant therapies, a signifi- cant proportion of patients presents with recurrence; moreover, patients with the same tumor stages may show different out- comes, indicating that the conventional staging procedures may be unable to precisely predict cancer prognosis (7, 8). Therefore, the search for new prognostic factors capable of identifying high-risk patients and of modulating cancer treatment options is still actively ongoing (9). In this regard, many studies have focused on innovative molecular markers playing an important role in cell cycle regulation, apoptosis, and tumor neoangiogen- esis. Uncontrolled cell proliferation is the hallmark of cancer, and there is increasing evidence that tumor cells have a damaged cell cycle-regulatory machinery. p27, an inhibitor of cyclin- dependent kinases that regulates the G 1 -S phase by blocking the cell cycle and maintaining cells in resting state (G 0 ), seems to plays an important role in the negative regulation of cell growth (10 –12). Low or absent p27 protein expression has been de- scribed in the cells of a large variety of human tumors, including CRCs (3, 13). However, contrasting results have been reported on the association between low or absent p27 protein and both survival and recurrence rate (12, 14). Thus, the consequence of low or high p27 protein expression in CRCs still remains un- clear. The p53 tumor suppressor gene has various important functions in cellular integration, including response to DNA damage, regulation of transcription, and control of genomic stability. In addition to cyclins, p53 influences the G 1 -S-phase checkpoint, and plays a pivotal role in apoptosis (15). The loss of the cell cycle apoptotic control mechanism through p53 mutation is believed to be one of the most important mecha- nisms of tumorigenesis in many human tumors (16). Mutations in the p53 gene with overexpression of its protein product are present in up to 70% of CRCs and have been proposed to be a late event in the progression from adenoma to colon carcinoma, after APC and K-ras mutations (17). A correlation between p53 alteration and poor prognosis has been demonstrated in many studies (3, 18); however, some investigations have reported Received 6/26/03; revised 1/23/04; accepted 2/16/04. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Gennaro Galizia, Second University of Naples School of Medicine, “F. Magrassi - A. Lanzara” Department of Clinical and Experimental Medicine and Surgery, c/o II Policlinico, Edificio 17, Via Pansini, 5, 80131 Naples, Italy. Phone: 39-081-566-6613; Fax: 39-081-566-6817; E-mail: gennaro.galizia@unina2.it. 3490 Vol. 10, 3490 –3499, May 15, 2004 Clinical Cancer Research Downloaded from http://aacrjournals.org/clincancerres/article-pdf/10/10/3490/1952257/zdf01004003490.pdf by guest on 21 June 2022